The long-term goal of this project is to elucidate molecular mechanisms controlling the number and function of nicotinic acetylcholine receptors (AChRs) on neurons. AChR genes expressed in chick ciliary ganglion neurons will be identified, cloned, and inserted into vectors for transfection of COS cells and chick primary fibroblasts, myotubes, and neurons. Subunit- specific mono- and polyclonal antibodies generated against fusion proteins will be used to examine AChR assembly accumulation in transfected cells. Whole cell patch clamp and single channel recording will be used to examine functional properties of the assembled receptors. Assembled receptors will be correlated with AChR subtypes expressed in ciliary ganglion neurons. Second messenger effects on AChR assembly and function will first be identified in ciliary ganglion neurons, and then analyzed for mechanism in transfected cells. Of special interest will be the process by which cyclic AMP increases the ACh response of ciliary ganglion neurons by seeming to convert AChRs from a "silent" state to one that is "functionally available". Subunit substitution and site-directed mutagenesis will be used to identify receptor loci necessary for the regulation. Mammalian neuronal AChRs will be examined for similar types of second messenger regulation to evaluate the generality of the findings and to identify possible differences along with their molecular bases. The genetics of Drosophila melanogaster will be used to examine the function and regulation of neuronal AChRs in vivo. Drosophila AChR genes will be identified and cloned. Mutants will be obtained by P element-mediated insertional mutagenesis and by mutagenizing chromosomes over a deficiency for the locus. Conditional mutants will be used to identify second-site suppressor mutations that may represent non-AChR genes which interact with or regulate AChRs to influence their number and functional state. Results obtained will provide information about regulatory mechanisms controlling a key postsynaptic component required for synaptic transmission. Second messenger regulation of neurotransmitter receptors provides in principle a mechanism for modulating synaptic function in a reversible manner responsive to cell-cell interactions.